PROJECT SUMMARY The broad ongoing objective of this research program is to gain a deeper understanding of the fundamental mechanisms by which the focal adhesion adaptor/scaffold proteins paxillin and Hic-5 contribute to cell-matrix signaling and thereby control of cell polarity and migration in development and disease. Importantly, communication between the three elements of the cytoskeleton, the trafficking machinery and cell-matrix interactions is essential for establishing both apical-basal and front-rear migration polarity. However, our understanding of the key mechanisms coordinating and integrating these processes remains incomplete. Through our development of new paxillin and Hic-5 knock out mouse models, in combination with the use of various ex-vivo 1D, 2D and 3D cell matrix and organoid model systems and real time imaging approaches, we have identified new roles for paxillin in establishing epithelial and mesenchymal cell polarity, including regulation of centrosome and Golgi organization and microtubule stability via control of HDAC6 activity. We have also identified Hic-5 as a new mediator of F-actin-intermediate filament cross talk, mechanobiology and 3D extracellular matrix deposition and remodeling. Using these model systems in conjunction with quantitative real time confocal, light sheet and super resolution imaging approaches, the main goals that we will address in the upcoming funding period are- Goal 1: How does paxillin contribute to the regulation of polarized trafficking in directed mesenchymal cell migration? Goal 2: What is the role of paxillin in establishment of apical-basal polarity and in branching morphogenesis in mammary epithelial cells and Goal 3: How does Hic-5 regulate the vimentin intermediate filament cytoskeleton organization and function in motile cells and during epithelial-mesenchymal transition? Through the elucidation of these mechanisms we will be better positioned to develop rational approaches to disease intervention and to appreciate the basis of developmental disorders.